The present invention relates to peptide chemistry as it applies to pharmaceutical research and development. The invention provides individual tetragonal flat rod shaped or plate-like crystals of glucagon-like peptide-1 related molecules, processes for their preparation, compositions and uses for these improved crystal forms.
GLP-1, a 37 amino acid peptide naturally formed by proteolysis of the 160 amino acid precursor protein preproglucagon, was first identified in 1987 as an incretin hormone. GLP-1 is secreted by the L-cells of the intestine in response to food ingestion and has been found to stimulate insulin secretion (insulinotropic action) causing glucose uptake by cells which decreases serum glucose levels (see, e g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)). GLP-1 is poorly active. A subsequent endogenous cleavage between the 6th and 7th position produces a more potent biologically active GLP-1(7-37)OH peptide. Approximately 80% of the GLP-1(7-37)OH so produced is amidated at the C-terminal in conjunction with removal of the terminal glycine residue in the L-cells and is commonly referred to GLP-1(7-36)NH2. Molecules which are reasonably homologous to, or are derived from, or based on these native forms will generally be referred to as GLP""s in this specification.
The biological effects and metabolic turnover of the free acid, the amide form, and many of the numerous known GLP""s are similar and show promise as agents for the treatment of diabetes, obesity, and related conditions, including but not limited to impaired glucose tolerance and insulin resistance. However, many GLP""s suffer from extremely short biological half lives, some as short as 3-5 minutes, which makes them unattractive for use as pharmaceutical agents. Presently, the activity of dipeptidyl-peptidase-IV (DPP-IV) is believed to readily inactivate many GLP""s and is in part responsible for the very short serum half lives observed. Rapid absorption and clearance following parenteral administration are also factors. Thus, there is a need to find a means for prolonging the action of these promising agents.
One such approach has been to modify these molecules to protect them from in vivo cleavage by DPP-IV. For example, see U.S. Pat. No. 5,512,549. In the insulin arts, it has long been known that extended time action can be achieved by administering crystalline protein formulations into the subcutis which act like depots, paying out soluble protein over time.
Heterogeneous micro crystalline clusters of GLP-1(7-37)OH have been grown from saline solutions and examined after crystal soaking treatment with zinc and/or m-cresol (Kim and Haren, Pharma. Res. Vol. 12 No. 11 (1995)). Also, crude crystalline suspensions of GLP(7-36)NH2 containing needle-like crystals and amorphous precipitation have been prepared from phosphate solutions containing zinc or protamine (Pridal, et. al., International Journal of Pharmaceutics Vol. 136, pp. 53-59 (1996)). Also, EP 0 619 322 A2 describes the preparation of micro-crystalline forms of GLP-1(7-37)OH by mixing solutions of the protein in pH 7-8.5 buffer with certain combinations of salts and low molecular weight polyethylene glycols (PEG). However, such crystalline clusters and crude suspensions are less than ideal for preparing long acting pharmaceutical formulations of GLP""s since they are loosely bound heterogeneous clusters of crystals or amorphous-crystalline suspensions which tend to trap impurities and are otherwise difficult to reproducibly manufacture and administer.
Most unexpectedly it was discovered that single tetragonal flat rod shaped or plate-like crystals of various GLP""s could be reproducibly formed from a mother liquor containing a GLP dissolved in a buffered solution and a C1-3 alcohol, or optionally a mono or disaccharide, over a wide range of pH conditions. The resulting single flat rod shaped or plate-like crystals are superior to, and offer significant advantages over, the GLP-1(7-37)OH crystal clusters or crude suspensions known in the art.
The single tetragonal flat rod shaped or plate-like crystals of the present invention are less prone to trap impurities and therefore may be produced in greater yields and administered more reproducibly than the known heterogeneous clusters. The crystal compositions of the present invention are pharmaceutically attractive because they are relatively uniform and remain in suspension for a longer period of time than the crystalline clusters or amorphous crystalline suspensions which tend to settle rapidly, aggregate or clump together, clog syringe needles and generally exacerbate unpredictable dosing. Most importantly, the crystal compositions of the present invention display extended, uniform, and reproducible pharmacokinetics which can be modulated by adding zinc using conventional crystal soaking techniques or, alternatively, by including zinc in the crystallization solution.
The present invention includes processes for preparing single rod-shaped or plate-like crystals of glucagon-like peptide-1 related molecules (GLP""s) which comprises preparing a crystallization solution comprising a purified GLP, a buffering agent containing an alcohol or a mono or di saccharide, and optionally, ammonium sulfate or zinc. In another embodiment the GLP crystals having tetragonal flat rod shaped or plate-like morphology selected from the group consisting of a GLP-1 analog, a GLP-1 derivative, a DPP-IV protected GLP, a GLP-1 peptide analog, or a biosynthetic GLP-1 analog are claimed. The invention also includes substantially homogenous compositions of GLP crystals, pharmaceutical formulations and processes for preparing such formulations, and methods for treating diabetes, obesity and related conditions.
By custom in the art, the amino terminus of GLP-1(7-37)OH has been assigned number residue 7 and the carboxy-terminus, number 37. This nomenclature carries over to other GLP""s. When not specified, the C-terminal is usually considered to be in the traditional carboxyl form. The amino acid sequence and preparation of GLP-1(7-37)OH is well-known in the art. See U.S. Pat. No. 5,120,712, the teachings of which are herein incorporated by reference. For the convenience of the reader the sequence is provided below.
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-COOH (SEQ ID NO:1)
xe2x80x9cGLP-1 analogxe2x80x9d is defined as a molecule having one or more amino acid substitutions, deletions, inversions, or additions relative to GLP-1(7-37) and may include the d-amino acid forms. Numerous GLP-1 analogs are known in the art and include, but are not limited to, GLP-1(7-34), GLP-1(7-35), GLP-1(7-36)NH2, Gln9-GLP-1(7-37), d-Gln9-GLP-1(7-37), Thr16-Lys18-GLP-1(7-37), and Lys18-GLP-1(7-37), Gly8-GLP-1(7-36)NH2, Gly8-GLP-1(7-37)OH, Val8-GLP-1(7-37)OH, Met8-GLP-1(7-37)OH, acetyl-Lys9-GLP-1(7-37), Thr9-GLP-1(7-37), D-Thr9-GLP-1(7-37), Asn9-GLP-1(7-37), D-Asn9-GLP-1(7-37), Ser22-Arg23-Arg24-Gln26-GLP-l(7-37), Arg23-GLP-1(7-37), Arg24-GLP-1(7-37), xcex1-methyl-Ala8-GLP-1(7-36)NH2, and Gly8-Gln21-GLP-1(7-37)OH, and the like.
Other GLP-1 analogs consistent with the present invention are described by the formula:
R1-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R2 (SEQ ID NO:2) wherein: R1 is selected from the group consisting of L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, beta-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine; X is selected from the group consisting of Ala, Gly, Val, Thr, Met, Ile, and alpha-methyl-Ala; Y is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; and R2 is selected from the group consisting of NH2, and Gly-OH.
GLP-1 analogs have also been described in WO 91/11457, and include GLP-1(7-34), GLP-1(7-35), GLP-1(7-36), or GLP-1(7-37), or the amide form thereof, and pharmaceutically-acceptable salts thereof, having at least one modification selected from the group consisting of:
(a) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine, or D-lysine for lysine at position 26 and/or position 34; or substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, lysine, or a D-arginine for arginine at position 36;
(b) substitution of an oxidation-resistant amino acid for tryptophan at position 31;
(c) substitution of at least one of: tyrosine for valine at position 16; lysine for serine at position 18; aspartic acid for glutamic acid at position 21; serine for glycine at position 22; arginine for glutamine at position 23; arginine for alanine at position 24; and glutamine for lysine at position 26; and
(d) substitution of at least one of: glycine, serine, or cysteine for alanine at position 8; aspartic acid, glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glutamic acid at position 9; serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glycine at position 10; and glutamic acid for aspartic acid at position 15; and
(e) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine, or the D- or N-acylated or alkylated form of histidine for histidine at position 7; wherein, in the substitutions in (a), (b), (d), and (e), the substituted amino acids can optionally be in the D-form and the amino acids substituted at position 7 can optionally be in the N-acylated or N-alkylated form.
A xe2x80x9cGLP-1 derivativexe2x80x9d is defined as a molecule having the amino acid sequence of GLP-1(7-37) or of a GLP-1 analog, but additionally having chemical modification of one or more of its amino acid side groups, xcex1-carbon atoms, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at amino acid side groups include, without limitation, acylation of lysine xcex5-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the terminal amino include, without limitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. Furthermore, one or more side groups, or terminal groups, may be protected by protective groups known to the ordinarily-skilled protein chemist. The xcex1-carbon of an amino acid may be mono- or dimethylated.
Other GLP-1 derivatives are claimed in U.S. Pat. No. 5,188,666, which is expressly incorporated by reference. Such molecules are selected from the group consisting of a peptide having the amino acid sequence:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-X (SEQ ID NO:3)
and pharmaceutically-acceptable salts thereof, wherein X is selected from the group consisting of Lys-COOH and Lys-Gly-COOH; and a derivative of said peptide, wherein said peptide is selected from the group consisting of: a pharmaceutically-acceptable lower alkyl ester of said peptide; and a pharmaceutically-acceptable amide of said peptide selected from the group consisting of amide, lower alkyl amide, and lower dialkyl amide.
Yet other GLP-1 derivatives consistent for use in the present invention include compounds claimed in U.S. Pat. No. 5,512,549, which is expressly incorporated herein by reference, described by the formula: 
wherein R1 is selected from the group consisting of 4-imidazopropionyl, 4-imidazoacetyl, or 4-imidazo-xcex1, xcex1 dimethyl-acetyl; R2 is selected from the group consisting of C6-C10 unbranched acyl, or is absent; R3 is selected from the group consisting of Gly-OH or NH2; and, Xaa is Lys or Arg, may be used in present invention.
xe2x80x9cDPP-IV protected GLP""sxe2x80x9d refers to GLP-1 analogs which are resistant to the action of DPP-IV. These include analogs having a modified or d amino acid residue in position 8. These also include biosynthetic GLP-1 analogs having Gly or the 1 amino acid residues Val, Thr, Met, Ser, Cys, or Asp in position 8. Other DPP-IV protected GLP""s include des amino His7 derivatives. xe2x80x9cGLP-1 peptide analogsxe2x80x9d are defined as GLP-1 analogs or derivatives which exclude acylated forms. xe2x80x9cBiosynthetic GLP-1 analogsxe2x80x9d are defined as any GLP-1 analogs or derivatives which contain only naturally occurring amino acid residues and are thus capable of being expressed by living cells, including recombinant cells and organisms. xe2x80x9cTreatingxe2x80x9d is defined as the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. Treating diabetes therefore includes the maintenance of physiologically desirable blood glucose levels in patients in need thereof.
The flat rod shaped or plate-like GLP crystals of the present invention, which are prepared using the claimed process, vary in size and shape to some degree. Generally, they range in size from approximately 2-25 microns (xcexcm) by 10-150 xcexcm and are flat, having a depth of approximately 0.5-5 xcexcm. These single crystals form from a single nucleation point and do not appear as multiple spiked star-like clusters known in the art.
Given the sequence information herein disclosed and the state of the art in solid phase protein synthesis, GLP""s can be obtained via chemical synthesis. However, it also is possible to obtain some GLP""s by enzymatically fragmenting proglucagon using techniques well known to the artisan. Moreover, well known recombinant DNA techniques may be used to express GLP""s consistent with the invention.
The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area such as Dugas, H. and Penney, C., Bioorganic Chemistry (1981) Springer-Verlag, New York, pgs. 54-92, Merrifield, J. M., Chem. Soc., 85:2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis, pp. 24-66, Freeman (San Francisco, 1969).
Likewise, the state of the art in molecular biology provides the ordinarily skilled artisan another means by which GLP""s can be obtained. Although GLP""s may be produced by solid phase peptide synthesis, recombinant methods, or by fragmenting glucagon, recombinant methods are preferable when producing biosynthetic GLP-1 analogs because higher yields are possible.
For purposes of the present invention, GLP-1 peptide analogs and biosynthetic GLP-1 peptide analogs are preferred. More preferred are the DPP-IV protected GLP""s, More highly preferred are biosynthetic GLP-1 peptide analogs. Another preferred group of GLP-1 peptide analogs are those which contain a single amino acid substitution at the 8 position which may include d and modified amino acid residues. More highly preferred biosynthetic GLP-1 peptide analogs are those which contain a single amino acid substitution at the 8 position, more preferably those which contain Gly or the 1 amino acid residues Val, Thr or Met in the 8 position.
The present invention provides a process for producing individual tetragonal rod shaped GLP crystals from a mother liquor. Under low to neutral pH conditions ranging from about pH 6-7, preferably about 6.4xc2x1about 0.2, the crystallization solution, or mother liquor, contains a final GLP concentration of about 1-10 mg/ml, preferably 2-7 mg/ml.
A number of conventional buffer solutions containing an alcohol or mono or disaccharide are suitable in the practice of the invention. 10 to 50 mM Tris, ammonium acetate, sodium acetate, or Bis-Tris is preferred. The concentration of alcohol ranges from about 2-15% (v/v), preferably 3-13%. Preferred alcohols are selected from the group containing methanol, ethanol, propanol, or glycerol, ethanol being most preferred.
Optionally, the addition of approximately 1% (w/v) ammonium sulfate to the mother liquor will generally increase the yield of crystals. The skilled artisan will also recognize the benefits of adding a preservative such as sodium azide and other such preservatives to the mother liquor to prevent bacterial growth.
In another embodiment, mono or disaccharides may be substituted for the alcohol in the same ratios on a weight to volume basis. Mono or disaccharides suitable for use in the presently claimed process include trehalose, mannitol, glucose, erythrose, ribose, galactose, fructose, maltose, sucrose, and lactose, though trehalose is preferred.
In yet another embodiment of the present invention, the process may be carried out in a neutral or high pH, zinc-containing environment ranging from about pH 7-10, preferably about pH 7.2-9.7. Under these conditions, the GLP concentration is in the range of approximately 1-20 mg/ml, preferably about 2-10 mg/ml. Total zinc, in a molar ratio to GLP, ranges from about 0.5-1.7, preferably 0.6-1.5.
Under such neutral or high pH conditions with zinc, suitable buffers and salts range in concentration from about 10-100 mM glycine and 0-200 mM NaCl, preferably 40-60 mM glycine and 0-150 mM NaCl. Preferred buffers are glycine, aspartic acid and Tris. The alcohol or sugar conditions are as stated previously.
Once the mother liquor is prepared, it is allowed to stand at approximately 15-37xc2x0 C., preferably about 18-25xc2x0 C., for 12-48 hours until crystallization occurs. The crystals may then be transferred or otherwise handled without any noticeable deleterious effects to the crystalline morphology suggesting that such crystals may be stored for prolonged periods without suffering structural damage.
In another embodiment, a pharmaceutical formulation may be prepared by adding pharmaceutically acceptable excipients, carriers, preservatives, and diluents directly to the mother liquor after the cystals have formed. In this embodiment, crystallization and subsequent additions are performed under sterile conditions. Zinc may be added directly to the mother liquor to effect the incorporation of zinc into the crystals. Preservatives may be added to the mother liquor to provide formulations of crystals suitable for multiple injections from the same container. Other excipients, such as antioxidants, buffers, acids and bases for pH adjustments, isotonicity agents and the like, may also be added directly to the mother liquor after the crystals have formed.
In another embodiment, the invention provides homogenous compositions of individual tetragonal flat rod shaped or plate-like crystal of GLP""s. Prior to the processes herein disclosed and claimed, such compositions could not be achieved. The compositions of the invention are useful in manufacturing processes and for preparing pharmaceutical formulations having extended time action for the treatment or prevention of diabetes, obesity and related conditions.
The claimed GLP crystals and compositions may optionally be treated with zinc using conventional crystal soaking techniques. By soaking the crystals in about a 0.5 mg/ml solution of zinc, complexes of crystals are formed which serve to extend the time action of the administered GLP. Also, by varying the zinc concentration, the complex composition can be altered leading to longer or shorter time actions.
As noted the invention provides pharmaceutical formulations, which are comprised of single tetragonal flat rod shaped or plate-like crystal of a GLP, together with one or more pharmaceutically acceptable diluents, carriers, or excipients. The crystals can be formulated for parenteral administration for the therapeutic or prophylactic treatment of diabetes, obesity or related conditions. For example, the crystals of the present invention can be admixed with conventional pharmaceutical carriers and excipients. The formulations comprising the claimed crystals contain from about 0.5 to 50 mg/ml of the active GLP, and more specifically from about 1.0 to 10 mg/ml. Furthermore, the crystals of the present invention may be administered alone or in combination with other antidiabetic agents. For subcutaneous or intramuscular preparations, a sterile formulation of the crystals of the present invention can be administered as a suspension in the original or modified crystallization mother liquor or in a pharmaceutical diluent such as pyrogen-free distilled water, physiological saline, or 5% glucose solution. A suitable formulation of the crystals of the present invention may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g., an ester of a long-chain fatty acid such as ethyl oleate.
Pharmaceutically acceptable preservatives such as an alkylparaben, particularly methylparaben, ethylparaben, propylparaben, or butylparaben or chlorobutanol, phenol or meta-cresol are preferably added to the formulation to allow multi-dose use.
The formulation may also contain an isotonicity agent, which is an agent that is tolerated physiologically and imparts a suitable tonicity to the formulation to prevent the net flow of water across the cell membrane. Compounds, such as glycerin, are commonly used for such purposes at known concentrations. Other possible isotonicity agents include salts, e.g., NaCl, dextrose, mannitol, and lactose. Glycerin is the preferred isotonicity agent. The concentration of the isotonicity agent is in the range known in the art for parenteral formulations, and for glycerin, is preferably about 16 mg/mL to about 25 mg/mL.
The formulation may also contain a pharmaceutically acceptable buffering agent to control the pH at a desired level. The pH is ideally such as to be acceptable to the patient upon administration, yet one at which the formulation is sufficiently stable, both physically and chemically. Preferably, the pH is controlled from a mildly acidic pH to a mildly basic pH, such as, between about pH 5 and pH 9. More preferably, the pH is between about pH 6 and pH 8. Buffering agents include but are not limited to citrate, acetate, phosphate, Tris, or a basic amino acid, such as, lysine or arginine, which are known to be pharmaceutically acceptable in these pH ranges. Other pharmacologically acceptable buffers for buffering at pH in these ranges are known in the art. The selection and concentration of buffer is well within the skill of the art.